Water Research 178 (2020) 115814

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Water Research
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Viral, bacterial, and protozoan pathogens and fecal markers in wells

supplying groundwater to public water systems in Minnesota, USA
Joel P. Stokdyk a, Aaron D. Firnstahl a, James F. Walsh b, Susan K. Spencer c,
Jane R. de Lambert b, Anita C. Anderson b, Lih-In W. Rezania b, Burney A. Kieke Jr. d,
Mark A. Borchardt c, *
a
U.S. Geological Survey Upper Midwest Water Science Center, 2615 Yellowstone Drive, Marshfield, WI, 54449, United States
b
Minnesota Department of Health, 625 Robert St. N, St. Paul, MN, 55164, United States
c
U.S. Department of Agriculture-Agricultural Research Service, Environmentally Integrated Dairy Management Research Unit, 2615 Yellowstone Drive,
Marshfield, WI, 54449, United States
d
Marshfield Clinic Research Institute, 1000 N. Oak Ave, Marshfield, WI, 54449, United States

a r t i c l e i n f o a b s t r a c t

Article history: Drinking water supply wells can be contaminated by a broad range of waterborne pathogens. However,
Received 9 December 2019 groundwater assessments frequently measure microbial indicators or a single pathogen type, which
Received in revised form provides a limited characterization of potential health risk. This study assessed contamination of wells by
25 March 2020
testing for viral, bacterial, and protozoan pathogens and fecal markers. Wells supplying groundwater to
Accepted 8 April 2020
Available online 12 April 2020
community and noncommunity public water systems in Minnesota, USA (n ¼ 145) were sampled every
other month over one or two years and tested using 23 qPCR assays. Eighteen genetic targets were
detected at least once, and microbiological contamination was widespread (96% of 145 wells, 58% of 964
Keywords:
Groundwater
samples). The sewage-associated microbial indicators HF183 and pepper mild mottle virus were detected
Pathogens frequently. Human or zoonotic pathogens were detected in 70% of wells and 21% of samples by qPCR,
HF183 Bacteroides with Salmonella and Cryptosporidium detected more often than viruses. Samples positive by qPCR for
Pepper mild mottle virus adenovirus (HAdV), enterovirus, or Salmonella were analyzed by culture and for genotype or serotype.
Microbial indicators qPCR-positive Giardia and Cryptosporidium samples were analyzed by immunofluorescent assay (IFA),
Pathogen-indicator ratios and IFA and qPCR concentrations were correlated. Comparisons of indicator and pathogen occurrence at
the time of sampling showed that total coliforms, HF183, and Bacteroidales-like HumM2 had high
specificity and negative predictive values but generally low sensitivity and positive predictive values.
Pathogen-HF183 ratios in sewage have been used to estimate health risks from HF183 concentrations in
surface water, but in our groundwater samples Cryptosporidium oocyst:HF183 and HAdV:HF183 ratios
were approximately 10,000 times higher than ratios reported for sewage. qPCR measurements provided
a robust characterization of microbiological water quality, but interpretation of qPCR data in a regulatory
context is challenging because few studies link qPCR measurements to health risk.
Published by Elsevier Ltd.

1. Introduction coliform absence and enteric pathogenic virus absence provides a

Public water systems regulated under the United States Envi-
ronmental Protection Agency’s (USEPA) Ground Water Rule are
monitored by analysis of total coliforms following the Revised Total
Coliform Rule (USEPA, 2006a). The strong association between total
* Corresponding author. USDA-ARS, 2615 Yellowstone Drive, Marshfield, WI,
54449, USA.
E-mail address: Mark.Borchardt@usda.gov (M.A. Borchardt).
reasonable assurance that health risk is low when total coliform
tests are consistently negative (Fout et al., 2017). When total coli-
form is detected, follow-up tests aim to identify the sanitary defect
causing contamination (USEPA, 2013). However, the implications of
total coliform-positive samples are ambiguous in terms of path-
ogen presence (Fout et al., 2017). Though practical within the “find-
and-fix” approach of the Revised Total Coliform Rule, total coliform
analysis provides a limited characterization of groundwater quality
in terms of the presence and types of pathogens that threaten
public health.

https://doi.org/10.1016/j.watres.2020.115814
0043-1354/Published by Elsevier Ltd.
2 J.P. Stokdyk et al. / Water Research 178 (2020) 115814
The body of scientific literature demonstrates groundwater
vulnerability to various pathogens (Hynds et al., 2014; Murphy
et al., 2017), but many studies have focused on human enteric vi-
ruses, leaving nonviral groundwater pathogens relatively under-
studied. Hynds et al. (2014) reported that 54% of pathogen records
for studies conducted in the USA included viruses while only 20%
and 15% included bacterial and protozoan pathogens, respectively.
Likewise, Murphy et al. (2017) identified 56 virus occurrence
studies worldwide compared to 26 studies that included bacterial
pathogens and 10 studies that included protozoan pathogens.
While less frequently measured in groundwater, bacteria and pro-
tozoa represent a public health concern (Craun et al., 2010). For
example, Escherichia coli O157:H7 and Campylobacter jejuni caused
groundwater-borne outbreaks in Canada, New Zealand, and
Nebraska (Bartholomew et al., 2014; O’Connor, 2002; Pedati et al.,
2019). Understanding the range of pathogens capable of contami-
nating groundwater is important for assessing risk (Murphy et al.,
2017).
Pathogen presence is of special concern for the 80% of
noncommunity public water systems in the United States that
distribute nondisinfected water (USEPA, 2006c), but studies more
commonly focus on municipal or community wells (Hynds et al.,
2014). Noncommunity public water systems, which are systems
that provide water to locations outside consumers’ residences (e.g.,
schools, restaurants), are subject to less stringent monitoring than
community systems and have more exemptions available (USEPA,
2006a, 2013). For example, the Revised Total Coliform Rule re-
quires noncommunity groundwater systems serving 1,000 or fewer
people to sample less frequently than similarly sized community
systems (USEPA, 2013). Though noncommunity systems may serve
a small or changing population, they are an important potential
exposure route to groundwater-borne pathogens. For example,
noncommunity groundwater systems in the United States are
associated with many outbreaks (Craun et al., 2010; Wallender
et al., 2014). Moreover, the etiological agent is unknown for a
large proportion of these outbreaks (Craun et al., 2010; Wallender
et al., 2014).
Assessments of groundwater quality that identify pathogens can
inform strategies that reduce contamination or exposure (Craun
et al., 2006). Quantitative polymerase chain reaction (qPCR) pro-
vides a means to efficiently test for pathogens, but the fundamental
approach of detecting genetic material rather than living organisms
remains a stumbling block to its use and interpretation (e.g., Stelma
and Wymer, 2012). Relationships between molecular data and
illness demonstrate the importance and relevance of qPCR mea-
surements. For example, Teunis et al. (2008) developed a rela-
tionship between qPCR measurements and probability of norovirus
infection using experimental data, and Thebault et al. (2013) linked
qPCR measurements to infection and illness using observational
data from foodborne norovirus outbreaks. Likewise, studies un-
derlying the Recreational Water Quality Criteria established a
relationship between qPCR measurements of Enterococci and
illness (USEPA, 2012a), and an epidemiological study of public
water systems linked qPCR virus measurements to acute gastro-
intestinal illness (Borchardt et al., 2012). Analysis of groundwater
by qPCR is therefore an effective way to characterize risk and assess
the microbiological quality of public water.
The primary objective of this study was to assess microbiological
contamination of public water supply wells in Minnesota, USA.
Community and noncommunity wells across the state were
repeatedly sampled over one or two years and tested for viral,
bacterial, and protozoan pathogens using qPCR. We used pathogen
data to assess the performance of conventional and emerging mi-
crobial indicators, and we examined groundwater contamination
relative to geology and system type. Finally, we compared qPCR
measurements to results from other microbiological detection
methods.
2. Methods
2.1. Study area and public water systems
Minnesota is located in north central USA and has a population
of 5.6 million. Climate is continental, with statewide average
annual temperature and precipitation of 4.7  C and 668 mm (NOAA,
2019). There are 6,737 public water supply systems in Minnesota,
including 968 community systems and 5,769 noncommunity sys-
tems (Minnesota Department of Health, 2018). The noncommunity
systems include 5,297 transient systems (e.g., hotels) and 488
nontransient systems (e.g., schools) (Minnesota Department of
Health, Minnesota Drinking Water Information System Database).
Groundwater is used by 96% of community systems and 99% of
noncommunity systems. Twenty-six percent of community sys-
tems and 98% of noncommunity systems do not disinfect water
(Minnesota Department of Health, Minnesota Drinking Water In-
formation System Database).
2.2. Study well descriptions
Well depth ranged from 6 to 192 m, and age, casing length, and
open interval length varied (Table S1). Fifty-seven percent of the
study wells draw from Quaternary glacial sand and gravel deposits;
other aquifers include sandstone, fractured crystalline rock, car-
bonate rock, and mixed sandstone, carbonate rocks, and shale
(Table S2).
Geologic sensitivity represents estimates for the vertical time of
travel for water or contaminants from the land surface to reach the
aquifer, and the estimates are based on the permeability and
thickness of the overlying material (Geologic Sensitivity Project
Workgroup, 1991). For example, aquifers that lack low-
permeability cover have geologic sensitivity ratings of very high
or high, while those protected by clay- or shale-rich sediments have
geologic sensitivity ratings of low or very low. The distribution of
study wells was similar to wells statewide in terms of geologic
sensitivity and aquifer type (Table S2).
2.3. Study design and well selection
One hundred forty-five wells in Minnesota, USA were sampled
every other month for one of two study phases (typically six
samples per well; Table S3). Wells (n ¼ 89) for the first phase (May
2014 through April 2015) were randomly selected from among the
nondisinfecting, year-round community and nontransient
noncommunity systems in Minnesota. For the second phase (May
2015 through May 2016), wells (n ¼ 85) were selected without
regard to disinfection status, and those potentially vulnerable to
contamination based on geologic setting, well construction, and
past monitoring were prioritized, including 29 wells from phase
one (typically sampled six times per phase for a total of 12). The
two-phase selection process produced a sample of wells that rep-
resented the aquifers and geology of wells statewide (Table S2).
Overall, 88 community wells and 57 noncommunity wells from 123
public water systems were sampled (Fig. 1).
2.4. Sample collection
Samples for pathogens and fecal markers (n ¼ 964) were
collected by dead-end ultrafiltration (Smith and Hill, 2009) using
Hemodialyzer Rexeed-25s filters (Asahi Kasei Medical MT Corp.,
Oita, Japan). Groundwater volume sampled ranged from 140 to
 J.P. Stokdyk et al. / Water Research 178 (2020) 115814 3

Fig. 1. Locations of community (blue dots) and noncommunity (orange dots) public water supply wells sampled in Minnesota, USA. (For interpretation of the references to colour in
this figure legend, the reader is referred to the Web version of this article.)

1783 L (mean, 728 L); all samples were collected prior to treatment
or disinfection. Filters were shipped on ice and processed within
72 h. Filter elution, eluate secondary concentration, nucleic acid
extraction, and reverse transcription are described in the Supple-
mentary Material.
Samples for total coliform and E. coli (n ¼ 925) were collected
after ultrafilter sample collection. To comply with laboratory hold
time requirements, some samples were not collected on the same
day as sampling for qPCR.
2.5. qPCR
Samples were tested for pathogens and fecal markers using 23
qPCR assays (Table S4). Assays targeting the ttr gene for Salmonella
and the Shiga toxin 1- and 2-producing bacteria were added after
the study was initiated, so 130 samples were not tested for them
(n ¼ 834 samples tested). qPCR was performed using a Light-
Cycler® 480 instrument (Roche Diagnostics, Mannheim, Germany);
primers and hydrolysis probes are described in Supplementary
Material. qPCR was performed in duplicate, and the average of
positive replicates is reported.
Lambda phage DNA was used to evaluate all samples for
4 J.P. Stokdyk et al. / Water Research 178 (2020) 115814
inhibition of qPCR, and hepatitis G virus RNA was used to evaluate
all samples for inhibition of reverse transcription-qPCR, as
described in Supplementary Material. Negative controls were
included at all processing steps (secondary concentration, nucleic
acid extraction, reverse transcription, and qPCR) and must exhibit
no fluorescence above the baseline (i.e., no Cq value) for data to be
acceptable. Modified live virus vaccines (Zoetis Inc., Kalamazoo, MI)
were used for DNA (bovine herpes virus) and RNA (bovine respi-
ratory syncytial virus) extraction positive controls, with the latter
serving also as the reverse transcription positive control. gBlocks®
and Ultramer® oligos (Integrated DNA Technologies, Coralville, IA)
were used as qPCR positive controls. Standard curve parameters
(Table S5), assay 95% LODs (limit of detection; Table S6), and results
for laboratory controls are reported in Supplementary Material.
2.6. Virus culture and genotyping
Adenovirus- and enterovirus-qPCR positive samples were
analyzed by cell culture and integrated cell culture-qPCR (ICC-
qPCR) and were genotyped using methods described by Borchardt
et al. (2012). Human adenoviruses (HAdV) were cultured in human
lung adenocarcinoma epithelial cells (A549) (CellPro Labs, Still-
water, MN) and enteroviruses in buffalo green monkey kidney cells
(BGMK) (CellPro Labs) (Fout et al., 1996; Lee et al., 2004). Cultures
were considered positive by exhibiting cytopathic effect or by
finding the qPCR-measured viral gene target concentration in cul-
ture was 10 times greater than that in the inoculum. Genotypes
were identified by sequencing, for HAdV, the 263 or 290 bp hexon
gene amplicon from the qPCR assay, and for enterovirus, the 656 bp
amplicon from a separate PCR for the region encoding 5’ UTR, VP4,
and VP2 (Ishiko et al., 2002). PCR products were visualized by gel
electrophoresis, cleaned (illustra GFX PCR DNA; GE Healthcare,
Buckingham, UK), purified (Gel Band Purification Kit; GE Health-
care) and sequenced with the BigDye Terminator Cycle Sequencing
Kit (Applied Biosystems, Foster City, CA). Consensus sequences
were constructed with Lasergene (DNAStar, Madison, WI) and
identified using BLAST (National Center for Biotechnology Infor-
mation, Bethesda, MD).
2.7. Salmonella culture, serotyping, and molecular subtyping
Salmonella-qPCR positive samples were cultured by pre-
enriching sample concentrate in peptone water and inoculating
into two selective broths, Rappaport Vassiliadis and tetrathionate.
After overnight incubation, each broth was streaked onto XLD agar
and brilliant green agar (BGA) plates. Presumptive Salmonella col-
onies were confirmed by qPCR; all confirmed colonies were posi-
tive by the invA and ttr gene assays. qPCR-confirmed isolates were
stored at 20  C until further analysis.
Salmonella serotype and molecular subtype were determined by
two methods: 1) agglutination profiles of isolates treated with a
panel of antisera specific for a variety of somatic (O) and flagellar
(H) antigens and 2) pulsed-field gel electrophoresis (PFGE) in which
an isolate’s electropherotype was linked to serotype using the
PulseNet database developed by the US Centers for Disease Control
and Prevention (Swaminathan et al., 2001). Salmonella culture,
serotyping, and molecular subtyping are further described in
Supplementary Material.
2.8. Direct immunofluorescent assay (IFA) for Giardia and
Cryptosporidium
Samples positive by qPCR for Giardia or Cryptosporidium were
microscopically examined for cysts and oocysts by IFA. Secondary
concentrate (100 mL) was treated with the Merifluor
Cryptosporidium/Giardia detection kit (Meridian Biosciences, Inc.,
Cincinnati, OH) as described in Stokdyk et al. (2019). Thirty
randomly selected qPCR-negative samples were also analyzed by
IFA.
2.9. Comparison of qPCR and IFA for Cryptosporidium and Giardia
Linear regressions of IFA and qPCR concentrations for Giardia
were completed using Microsoft Excel 2016. Two Giardia detections
lacked concentrations (due to unknown sample volume) and were
therefore excluded from the regressions, so 18 samples were
included. Separate regressions were completed using only those
samples positive by both methods (n ¼ 12). Linear regressions of
IFA and qPCR concentrations for Cryptosporidium are reported in
Stokdyk et al. (2019).
2.10. Total coliform and E. coli analysis
Samples for total coliform and E. coli were analyzed within 30 h
of sample collection following standard method 9223 (Rice et al.,
2012).
2.11. Microbial indicator performance
Sensitivity, specificity, and positive and negative predictive
value were derived from logistic regression models for the HF183/
BacR287 assay for human Bacteroides (hereafter HF183), Bacter-
oidales-like HumM2, pepper mild mottle virus (PMMoV), and total
coliforms relative to pathogen detections on a sample-level and
well-level following Fout et al. (2017). For the sample-level ana-
lyses, mixed logistic regression models with random intercepts for
well and a spatial correlation structure for the samples within a
well were fit to address the non-independent nature of the data
(Chavance and Escolano, 2016; Morel et al., 2003). For the well-
level analyses, the data were considered independent.
Population-averaged logistic regression models were fit with
adjustment for number of times the well was sampled (categorized
as 1e5, 6, or 7e12 times), since this could affect the corresponding
probability of pathogen detection for the well. Because no wells
sampled 7e12 times were negative for HF183, the adjustment for
HF183 combined wells sampled 7e12 times with wells sampled 6
times.
The strength of the indicator-pathogen association was also
quantified by calculating the pathogen detection ratio (positive
predictive value/[1-negative predictive value]). The pathogen
detection ratio is identical to the risk ratio calculated in Fout et al.
(2017), where values greater than 1.0 (the null) represent the
relative elevation in the pathogen detection rate if the indicator is
detected versus when the indicator is not detected. For example, a
pathogen detection ratio of 1.5 indicates that detection of a path-
ogen was 50% more likely when the indicator was detected
compared to when the indicator was not detected. Indicator per-
formance was assessed for any human or zoonotic pathogen
detection and the taxonomic subgroups therein: viral, bacterial,
and protozoan pathogens. Analyses were performed using SAS 9.4
(SAS Institute, Inc., Cary, NC).
3. Results
3.1. qPCR
Overall, 18 of the 23 qPCR genetic targets were detected in one
or more samples (Table 1), and 558 samples (58%) were positive for
at least one genetic target; 230 samples were positive for multiple
genetic targets. Adenovirus group B, hepatitis A virus, norovirus
 J.P. Stokdyk et al. / Water Research 178 (2020) 115814 5

Table 1
Number and concentrations of samples (n ¼ 964) positive for genetic targets by qPCR.

Organism type Microbial targeta No. positive samples (%)c Concentration of positive samples (gc L1)

Median Mean Maximum

Virus Adenovirus group A 3 (0.3) 0.007 3.8 11

Adenovirus groups C, D, F 21 (2.2) 1.1 42 638
Adenovirus (any group) 24 (2.5) 0.9 40 638
Human enterovirus 9 (0.9) 0.5 0.9 2.3
Human polyomavirus 11 (1.1) 0.2 0.4 1.6
Norovirus genogroup II 5 (0.5) 63 103 222
Bovine polyomavirus 14 (1.5) 0.5 1.6 6.4
Pepper mild mottle virus 52 (5.4) 8.9 42 920
Rotavirus group A 14 (1.5) 6.5 28 228
Virus totalb 110 (11.4) 4.1 31 920

Bacteria Bacteroidales-like HumM2 81 (8.4) 0.4 2.5 68

Human Bacteroides HF183 410 (43) 0.4 4.0 374
Bacteroidales-like Cow M3 1 (0.1) 2.3 2.3 2.3
Ruminant Bacteroides 47 (4.9) 0.8 2.8 47
Campylobacter jejuni 1 (0.1) d d d
Shiga toxin 2-producing bacteria 3 (0.4) 0.2 0.5 1.1
Salmonella (invA) 48 (5.1) 0.8 302 9538
Salmonella (ttr) 35 (4.3) 3.1 1810 38236
Salmonella (either gene target) 59 (6.1) 1.3 938 38236
Bacteria totalb 482 (50.0) 0.5 128 38236

Protozoa Cryptosporidium spp. 107 (11.1) 0.1 10 246

Giardia duodenalis assemblage B 20 (2.1) 0.7 3.1 16
Protozoa totalb 121 (13) 0.2 9.4 246

Any genetic targetb 558 (58) 0.54 97 38236

gc, gene copies.

a
Samples were negative for adenovirus group B, enteropathogenic E. coli, Shiga toxin 1-producing bacteria, hepatitis A virus, norovirus genogroup I.
b
The positive sample totals for virus, bacteria, protozoa and any genetic target are less than the sum of individual targets because some samples were positive for more than
one target.
c
n ¼ 964 except HF183 (n ¼ 949), Salmonella invA (n ¼ 950), Salmonella ttr (n ¼ 822), and Shiga toxin 2-producing bacteria (n ¼ 834).
d
C. jejuni concentration was not calculated because sample volume was unknown.

genogroup I, enteropathogenic E. coli, and Shiga toxin 1-producing
bacteria were not detected. HF183 was detected most frequently
(Table 1) and often alone (23% of samples). Bacteria other than
HF183 were detected in 18% of samples, more frequently than
protozoa and viruses. Human or zoonotic pathogens (all pathogens
listed in Table 1 excluding bovine polyomavirus) were detected in
207 samples (21%). Viral, bacterial, and protozoan pathogens were
detected in 55 (6%), 62 (6%), and 121 (13%) samples, respectively.
Thirty-nine samples (4%) were positive for multiple pathogens.
Concentrations were generally low, but eight genetic targets,
including six pathogens, had a maximum concentration greater
than 100 gene copies L1 (Table 1).
Wells were typically sampled six times each (range 1e12; see
Table S3). Of the 139 wells positive for a genetic target (Table 2),
most (n ¼ 121) had more than one positive sample (Table S7).
While HF183 was the only genetic target in many samples, most
wells positive for HF183 (all but six) were positive for another ge-
netic target during the study. Human or zoonotic pathogens were
detected in 102 wells (70%), including 46 (32%), 50 (34%), and 59
(41%) wells with viral, bacterial, and protozoan pathogens,
respectively. An examination of detections by well depth showed
no apparent trends (Table S8). Genetic targets were detected in 98%
of wells from community systems and 93% of wells from
noncommunity systems.
The 29 wells included in both study phases were sampled 7e12
times each, and 192 of the 333 samples from these wells were
positive for one or more genetic targets. All 29 of these wells had
two or more samples positive for a genetic target. Individual targets
were detected in these 29 wells at frequencies similar to the overall
results reported in Table 1. More samples were positive from these
29 wells in phase 2 (n ¼ 120 positive samples) than phase 1
(n ¼ 72).
Samples collected in phase two of the study were positive for
one or more genetic targets at a higher rate than samples collected
in phase one (75% and 41%, respectively), largely driven by the
bacteria HF183 (66% and 23% positive, respectively). The proportion
of wells positive for phase two (100%) was greater than for phase
one (91%), and the difference was driven primarily by HF183 (100%
and 80% positive, respectively).
Genetic targets specific to human fecal waste were detected in
47% of samples and 94% of wells and included HF183, Bacteroidales-
like HumM2, and the human viruses adenovirus, enterovirus, pol-
yomavirus, and norovirus GII. Genetic targets specific to ruminant/
bovine fecal waste, including ruminant Bacteroides, Bacteroidales-

Table 2
Number of community and noncommunity wells positive for viruses, bacteria, protozoa, and pathogens by qPCR.

Organism type Community (n ¼ 88) Noncommunity (n ¼ 57) All wells (n ¼ 145)

Virus 37 (42%) 35 (61%) 72 (50%)

Bacteria 84 (95%) 52 (91%) 136 (94%)
Protozoa 35 (40%) 24 (42%) 59 (41%)
Human or zoonotic pathogen 57 (65%) 45 (79%) 102 (70%)
Any genetic target 86 (98%) 53 (93%) 139 (96%)
6 J.P. Stokdyk et al. / Water Research 178 (2020) 115814
like Cow M3, and bovine polyomavirus, were detected in 6% of
samples and 34% of wells. Human-specific fecal microbes were
detected in 95% of community wells and 91% of noncommunity
wells. Ruminant/bovine-specific microbes were detected in 33% of
community wells and 35% of noncommunity wells. Eighty-three
percent of wells in phase one and 100% of wells in phase two
were positive for human-specific microbes, while 30% of wells in
phase one and 32% of wells in phase two were positive for bovine/
ruminant-specific microbes.
3.2. Virus cultures and genotyping
Human adenovirus culture was attempted with the 24 samples
positive for HAdV by qPCR. One cultured sample exhibited cyto-
pathic effect and five samples were positive by ICC-qPCR.
Sequencing revealed that 17 of the qPCR-positive samples were
HAdV 41, three were HAdV 5, three were HAdV 31, and one was
HAdV 6.
Human enterovirus culture of the nine qPCR-positive samples
showed all were negative by cytopathic effect and ICC-qPCR. Gen-
otyping was successful for six of the nine enterovirus samples;
three were coxsackievirus A6, one each was coxsackievirus B5,
echovirus 18, and echovirus 30.
3.3. Salmonella cultures and subtyping
Of the 59 samples positive for Salmonella by qPCR, seven were
positive on both XLD and BGA with an additional four positive on
XLD agar alone. Of the 11 culture-positive samples, subtyping
identified ten that were serovar Typhimurium and one serovar
Montevideo.
3.4. Giardia and Cryptosporidium immunofluorescent assay
Fourteen of 20 samples positive for Giardia by qPCR were pos-
itive by IFA at concentrations from <1 to 3.7 cysts L1; two de-
tections were qualitative due to missing sample volume. All seven
samples with qPCR concentrations >0.7 gene copies L1 were
positive by IFA, while only 5 of 11 below that level were positive by
IFA. Regression revealed a significant relationship between the
methods (R2 ¼ 0.94) and a ratio of 4.5 gene copies:cyst based on the
slope, though the regression included only 18 samples over a
limited range (Fig. 2). Regression restricted to samples positive by
Fig. 2. Relationship between Giardia concentrations for samples (n ¼ 18) measured by
quantitative polymerase chain reaction (qPCR) and immunofluorescent assay (IFA;
R2 ¼ 0.94, slope ¼ 4.5).
both methods (n ¼ 12) produced similar results (R2 ¼ 0.93,
slope ¼ 4.6).
Sixty-seven of 107 samples positive for Cryptosporidium by qPCR
were positive by IFA, with consistent IFA detections at qPCR con-
centrations >0.7 gene copies L1. Results for Cryptosporidium,
including qPCR-IFA regression and species identification and sub-
typing, are described in Stokdyk et al. (2019). Thirty of 30 randomly
selected qPCR-negative samples were negative by IFA for Crypto-
sporidium and Giardia.
3.5. Indicator performance
Total coliforms were detected in 53 of 925 samples and 36 of 145
wells. HF183, Bacteroidales-like HumM2, and PMMoV were detec-
ted in 133, 57, and 40 wells, respectively; all wells positive for
Bacteroidales-like HumM2 or PMMoV were also positive for HF183.
Sample-level detection frequencies are reported in Table 1; 57 and
30 samples positive for Bacteroidales-like HumM2 and PMMoV,
respectively, were also HF183-positive. Metrics for indicator per-
formance relative to any human or zoonotic pathogen are reported
in Table 3. Pathogen-indicator associations at the sample-level
were significantly elevated for HF183, Bacteroidales-like HumM2,
and coliforms, but at the well-level these associations were not
significantly elevated for any indicators. Restricting the assessment
to viral, bacterial, or protozoan pathogens resulted in the same
overall trend as observed for the all-pathogens analysis (Table S9).
E. coli was detected in three samples and performance metrics were
not calculated.
4. Discussion
4.1. Incidence of pathogens and microbial indicators
We completed a large, systematic study of community and
noncommunity public wells (n ¼ 145) across the geographic extent
of Minnesota, USA. Study wells represented diverse geologies, and
the two-year sampling campaign encompassed meteorological
conditions of all four seasons. Whereas previous groundwater
studies typically focused on microbial indicators or a single class of
pathogens, we included viral, bacterial, and protozoan pathogens
and fecal markers.
We observed widespread groundwater contamination (96% of
wells and 58% of samples) by a broad range of microorganisms.
Pathogens were detected in our study more frequently than studies
reviewed by Hynds et al. (2014), which reported 15% of samples and
16% of wells positive, but the reviewed studies typically included
fewer tested pathogens (an average of two to three) and fewer
samples per well (an average of 1.4). We sampled wells typically six
times, and while 102 wells (70%) were positive for human or zoo-
notic pathogens, only 49 wells (34%) were pathogen-positive more
than once. Likewise, of 207 pathogen-positive samples, only 39
(19%) were positive for more than one pathogen. While contami-
nation was widespread, the intermittence of well contamination
and variation in pathogens we observed are consistent with the
transient nature of groundwater contamination (e.g., Bradbury
et al., 2013).
Among pathogens, bacteria and protozoa are included in
groundwater studies less frequently than viruses (Hynds et al.,
2014; Murphy et al., 2017), but Salmonella and Cryptosporidium
were detected more frequently in our wells. In fact, Salmonella and
Cryptosporidium were detected in 33% and 40% of wells, respec-
tively, and they were the only pathogens present in 131 of the 207
pathogen-positive samples. Groundwater studies in developed
countries rarely include Salmonella outside of outbreak in-
vestigations (Levantesi et al., 2012; Murphy et al., 2017). Likewise,
 J.P. Stokdyk et al. / Water Research 178 (2020) 115814 7

Table 3
Sample- and well-level indicator test performance for indicating the presence of any human or zoonotic pathogen. Analyses were adjusted for well (sample-level) and number
of times a well was sampled (well-level). Values in parentheses are 95% confidence intervals.

Analysis Indicator Pathogen detection P- Sensitivityc Specificityd Positive predictive valuee Negative predictive valuef
level ratioa valueb (%) (%) (%) (%)

Sample HF183 n ¼ 949 1.84 <0.0001 57 (51e64) 61 (56e65) 26 (21e32) 86 (82e89)

Bacteroidales-like HumM2 1.72 0.009 11 (7e17) 94 (92e96) 32 (22e44) 81 (78e85)
n ¼ 964
Pepper mild mottle virus n ¼ 964 1.07 0.823 4 (2e7) 97 (95e98) 21 (11e35) 80 (77e84)
Total coliform n ¼ 925 1.91 0.003 5 (3e9) 98 (97e99) 36 (25e50) 81 (77e84)
Well HF183 n ¼ 145 1.26 0.158 92 (82e97) 22 (9e43) 85 (71e92) 33 (11e66)
Bacteroidales-like HumM2 1.04 0.539 33 (21e47) 73 (53e86) 85 (69e93) 19 (9e34)
n ¼ 145
Pepper mild mottle virus n ¼ 145 1.05 0.503 26 (16e39) 80 (60e91) 86 (68e94) 19 (10e34)
Total coliform n ¼ 145 1.04 0.642 27 (19e38) 77 (59e88) 85 (67e94) 18 (9e33)
a
Pathogen detection ratio ¼ positive predictive value/(1-negative predictive value). Values greater than 1.0 represent the relative elevation in the pathogen detection rate if
the indicator is detected versus when the indicator is not detected.
b
P-value for the t-test of association between the indicator and pathogen in a logistic regression model.
c
True positive rate: probability the indicator is detected given the pathogen is present.
d
True negative rate: probability the indicator is not detected given the pathogen is absent.
e
Probability the pathogen is detected given the indicator is present.
f
Probability the pathogen is not detected given the indicator is absent.

Cryptosporidium is not considered a contaminant of groundwater
unless surface water is entering wells. However, Cryptosporidium
was not associated with assessments of surface water influence in
our study wells (Stokdyk et al., 2019). Inclusion of nonviral patho-
gens is therefore important for characterizing groundwater-related
health risk.
The fecal markers HF183 and PMMoV were among the most
frequently detected organisms in our study wells, but their signif-
icance is difficult to assess because context for their incidence,
concentrations, and association with pathogens in groundwater is
scarce. Two studies reported incidence of HF183 in groundwater
(Allevi et al., 2013; Murphy et al., 2020), and reports of PMMoV in
wells are limited (Malla et al., 2019; Murphy et al., 2020; Symonds
et al., 2018). Moreover, pathogens were rarely measured in
conjunction with HF183 or PMMoV in groundwater (Murphy et al.,
2020; Symonds et al., 2018). While HF183 and PMMoV suggest fecal
contamination of these public wells, the implications for well
management and public health are unclear.
4.2. Contamination by system type, geology, precipitation, and
study phase
Despite potential differences in fecal sources, contaminant
transport factors, and water system characteristics, we observed
generally similar proportions of pathogens and fecal markers
among community and noncommunity wells. Noncommunity
wells were, however, pathogen-positive at a slightly higher rate,
which is of special concern because the majority in Minnesota
(98%) and the United States (80%) distribute nondisinfected water
(Minnesota Department of Health Drinking Water Information
System Database; USEPA, 2006c). Noncommunity systems are
linked to a large proportion of waterborne disease outbreaks and
report high rates of total coliform-positive samples compared to
community systems (Craun et al., 2010; Wallender et al., 2014;
USEPA, 2006c). However, the elevated risk apparently associated
with noncommunity groundwater systems is poorly defined: most
groundwater outbreaks in noncommunity systems have undeter-
mined etiology (Craun et al., 2010; USEPA, 2006c; Wallender et al.,
2014), and total coliforms provide a limited characterization of
water quality. Using qPCR, we observed a broad range of microbi-
ological contaminants in both noncommunity and community
wells.
Geology is among the factors related to microbiological
contamination of groundwater (Murphy et al., 2017), and studies
report groundwater vulnerability based on aquifer type (Allen et al.,
2017; Hynds et al., 2014). However, a high percentage of wells
(89e100%) in all five major aquifer types in Minnesota showed
microbiological contamination, and differences among aquifer
types appeared minor when viruses, bacteria, and protozoa were
considered as groups (Table 4). Likewise, geologic sensitivity
(Table 5), which reflects the travel time for contaminants from the
surface to reach groundwater, did not appear to follow microbio-
logical contamination. Comparisons among aquifer type are
tentative because three of five types had relatively few wells.
Rainfall influences groundwater contamination (Murphy et al.,
2017) because it is a driver of microbial transport (Allen et al.,
2017; Bradbury et al., 2013) and its low ionic strength facilitates
microbe detachment from sediment particles (Hunt and Johnson,
2017). Precipitation during both study phases exceeded Minneso-
ta’s historical average annual and included the wettest month on
record (NOAA, 2019), so elevated precipitation may have contrib-
uted to the widespread contamination of study wells. To the extent
that precipitation was a factor, contamination associated with
above-average rainfall may become less anomalous as the trend of
increasing precipitation in Minnesota is expected to continue
(Runkle et al., 2017).
The proportion of samples positive for a genetic target was
larger for phase two than phase one, which may reflect the targeted
selection of vulnerable wells for phase two. However, well selection
is confounded by temporal variation in factors related to microbial
source and transport, like precipitation and pathogen prevalence in
host populations. In fact, the 29 wells sampled in both phases were
also more often positive in phase two than one, suggesting that
variation over time, rather than well selection, contributed to dif-
ferences between study phases.
4.3. Microbial indicators of pathogen presence
Testing for total coliforms under the Revised Total Coliform Rule
forms the basis of microbial monitoring for public water systems
(USEPA, 2006a), and we used pathogen measurements to evaluate
its performance as a microbial indicator. Consistent with Fout et al.
(2017), at the sample level total coliform analysis was a good pre-
dictor of pathogen absence but a poor predictor of pathogen
presence, as demonstrated by high negative predictive value and
specificity but poor positive predictive value and sensitivity. For
8 J.P. Stokdyk et al. / Water Research 178 (2020) 115814
Table 4
Percentage of wells qPCR-positive for viruses, bacteria, and protozoa by aquifer type.
Organism type Sand and gravel Sandstone Fractured crystalline rock
(n ¼ 83) (n ¼ 33) (n ¼ 13)
Virus 40 70 54
Bacteria 92 97 100
Protozoa 47 21 54
Any genetic 94 100 100
target
Table 5
Percentage of wells qPCR-positive for viruses, bacteria, and protozoa by geologic sensitivity rating.
Organism type Very High (n ¼ 32) High (n ¼ 19)
Virus 38 42
Bacteria 97 95
Protozoa 50 37
Any genetic target 97 95
example, 98% of pathogen-negative samples were also negative for
total coliforms (specificity), but 95% of pathogen-positive samples
lacked the indicator (i.e., 5% sensitivity). At the well level the as-
sociation between coliforms and pathogens was not statistically
significant, and the performance metrics were ambiguous. Based
on these findings, a negative coliform test gives water managers
confidence that pathogens were also likely absent at the time of
sampling. However, the well could be pathogen-contaminated
later, which is particularly problematic for small public water sys-
tems required to test their wells only once per year.
Better indicators of pathogen presence are continually sought to
address the limitations of conventional ones. Human-associated
Bacteroides and PMMoV are becoming common indicators of hu-
man fecal pollution in water (Harwood et al., 2014; Korajkic et al.,
2018; Symonds et al., 2018), but, as with other microbial in-
dicators, their association with pathogens and health risk is a
source of uncertainty (Boehm et al., 2015; Harwood et al., 2014;
Korajkic et al., 2018). Our assessment demonstrated that HF183 and
PMMoV performed similar to total coliforms (Table 3) and other
indicators (Fout et al., 2017). The sample-level sensitivity of HF183
(57%) appears to suggest a better relationship with pathogens.
However, its low positive predictive value (26%) is consistent with
other indicators while its specificity (61%) is much worse, showing
that its elevated sensitivity is driven by ubiquitous incidence rather
than a stronger association with pathogens. Compared to sample-
level performance, well-level sensitivity and positive predictive
value increased at the expense of specificity and negative predictive
value for all indicators, which is consistent with trends described by
Fout et al. (2017).
Evaluation of indicator performance by pathogen detection ra-
tios showed that ratios for HF183, Bacteroidales-like HumM2, and
total coliform were significantly greater than 1.0 (the null value) at
the sample level; samples were 1.7e1.9 times more likely to have a
pathogen when these indicators were detected. However, only
26%e36% of samples positive for these indicators were positive for
a pathogen (i.e., positive predictive value), so the false-positive rate
was high. Pathogen risk ratios were not significantly greater than
1.0 for well-level analyses, signifying that indicator-positive and
indicator-negative wells had similar risks of being positive for a
pathogen.
Viral and bacterial indicators may more closely represent the
transport and survival of viral and bacterial pathogens, respectively
(e.g., Korajkic et al., 2018). However, the evaluation of indicators
relative to viral, bacterial, and protozoan pathogens showed that
HF183, Bacteroidales-like HumM2, and PMMoV were not better
Carbonate rocks Mixed sandstone, carbonate rocks, shale All
(n ¼ 9) (n ¼ 7) (n ¼ 145)
56 57 50
89 100 94
33 43 41
89 100 96
Moderate (n ¼ 33) Low (n ¼ 33) Very Low (n ¼ 28) All (n ¼ 145)
61 55 50 50
100 94 82 94
42 36 36 41
100 97 89 96
indicators of their analogous pathogens than of other pathogen
types (Table S9). Instead, the trends we observed in indicator per-
formance were generally consistent across indicators and pathogen
types. Only one groundwater study reports the incidence of HF183
with pathogens (Murphy et al., 2020), and the performance of
PMMoV as an indicator varied among the few studies in which it
was paired with pathogen measurements (Symonds et al., 2018).
We report the first robust assessment of HF183 and PMMoV per-
formance as indicators in groundwater and demonstrate that, like
other indicators (Fout et al., 2017), they reliably identify pathogen
absence but not presence.
4.4. qPCR microbial indicators and associated health risk
Health risks associated with HF183 and other fecal markers in
recreational water have been estimated by assuming the measured
concentration ratio between pathogens and indicators in sewage is
identical to the ratio in water (Ahmed et al., 2018; Boehm et al.
2015, 2018). For example, if the average concentration of HF183 in
sewage is ten times that of Cryptosporidium, then the unmeasured
Cryptosporidium concentration in recreational water is assumed to
be one-tenth of that measured for HF183; health risk is then esti-
mated by quantitative microbial risk assessment using the
extrapolated Cryptosporidium concentration. Improvements to the
ratio approach for estimating health risk from indicators account
for the differential decay of indicators and pathogens in the envi-
ronment (Boehm et al., 2018), underscoring that sewage ratios may
not represent ratios in the environment (i.e., where exposure oc-
curs), which a recent review demonstrates (Korajkic et al., 2018).
In our study, the pathogen-indicator concentrations measured
in public water system wells suggest even more caution is war-
ranted in extending the pathogen-indicator ratios in sewage to
estimate the health risk of indicator concentrations measured in
groundwater. For example, mean pathogen-indicator ratios for
Cryptosporidium and HAdV 40/41 relative to HF183 in raw sewage
were 5  105 and 5  104, respectively, and were used to estimate
exposure to these pathogens in recreational water (Ahmed et al.,
2018; Boehm et al., 2018) (see Supplemental Material for ratio
calculations). In contrast, mean ratios for our groundwater samples
were approximately 10,000 times higher, 0.32 oocysts:HF183
(based on IFA analysis for Cryptosporidium) and 8.8 HAdV 40/
41:HF183 (based on samples identified as HAdV 41 by genotyping).
Moreover, our mean ratios include HF183-positive samples that
were negative for these pathogens (i.e., ratio ¼ 0), and ratios varied
significantly among samples (coefficient of variation > 1000%). We
 J.P. Stokdyk et al. / Water Research 178 (2020) 115814
cannot estimate the health risk associated with HF183 in ground-
water by extrapolation of sewage-based ratios because they do not
accurately reflect exposure to groundwater-borne pathogens. Our
data suggest that these microbial indicators are useful for identi-
fying groundwater contamination in general, though not neces-
sarily pathogen presence or health risk.
While the health risk indicated by HF183 and PMMoV is un-
certain, the detection of pathogens in public wells represents a
potential health concern. To generally estimate the potential risk
associated with pathogen detections in our wells, we compared our
results to the relationship established by Borchardt et al. (2012) for
exposure to viruses in non-disinfected tap water supplied by public
wells. In the communities studied by Borchardt et al. (2012) the
relative risk of acute gastrointestinal illness among adults and
children increased 23% when the maximum concentration of vi-
ruses measured in the communities’ tap water exceeded 31 gene
copies L1. Nonviral pathogens were not considered and if present
would have likely increased risk further. Ten of our study wells had
maximum virus concentrations greater than 31 gene copies L1,
and water supplied by all ten wells was not disinfected, suggesting
the populations served had higher relative risk of illness. However,
none of these ten wells had indications of increased risk based on
HF183 or PMMoV. For example, the number of HF183- and PMMoV-
positive samples for these wells was similar to other wells, and the
mean concentration of samples positive for HF183 (1.2 gene copies
L1) and PMMoV (7.9 gene copies L1) in these wells was not
exceptional compared to samples overall (Table 1). In addition to
crudely estimating risk, this cursory comparison is consistent with
the low positive predictive values of HF183 and PMMoV and shows
the advantage of pathogen testing by qPCR.
4.5. Pathogen analysis by qPCR, culture, and IFA
To provide additional context for qPCR data, we analyzed five
pathogens by culture or IFA. Samples were positive by culture or IFA
across the range of qPCR concentrations for three of them
(adenovirus, Giardia, Cryptosporidium), and Giardia and Cryptospo-
ridium concentrations measured by IFA correlated well with qPCR
concentrations (Fig. 2; Stokdyk et al., 2019). However, samples for
all five of these organisms were less often culture- or IFA-positive at
low qPCR-measured concentrations (Table S10). Consistent with
our results, previous studies comparing measurements by qPCR
and other methods of the same environmental water samples show
that qPCR is more analytically sensitive (Borchardt et al., 2012;
Corsi et al., 2016; Stokdyk et al., 2019). Our data do not allow us to
differentiate the effects of false-negatives due to viable-but-not-
culturable organisms and qPCR detection of nonviable genetic
material. Nonetheless, the comparison shows concordance among
methods and offers context for interpreting molecular data,
including for dose-response relationships in quantitative microbial
risk assessments.
4.6. qPCR in regulatory settings
We observed widespread, transient contamination of wells by
diverse microorganisms, as well as disparities between pathogen
and indicator detections, which illustrate challenges that ground-
water regulators face. With advantages over microbial indicators
and other detection methods, qPCR pathogen analysis is a tool for
addressing these challenges. However, current groundwater regu-
lations in the United States lack a framework enabling regulators to
readily apply such data, and the use of pathogen measurements by
qPCR is often opposed or dismissed (e.g., Allen et al., 2000; Edberg
et al., 2000; Stelma and Wymer, 2012).
Despite the challenges with applying qPCR technology and
9
pathogen-specific measurements to groundwater management,
both are increasingly incorporated into federal regulation.
Pathogen-specific criteria are defined in Long Term 2 Enhanced
Surface Water Treatment Rule (USEPA, 2006b), and qPCR is
included in the Recreational Water Quality Criteria (USEPA, 2012a),
the Unregulated Contaminant Monitoring Regulation (USEPA,
2012b), and USEPA Methods 1615 (Fout et al., 2012) and 1696
(USEPA, 2019). More generally, studies that report the incidence of
specific pathogens are used to inform regulatory development (e.g.,
USEPA, 2006c). With growing ease of application to water testing,
qPCR offers a tool for groundwater research and management.
However, with few studies linking qPCR-measured concentrations
to health risk, regulators presented with PCR data for their public
water systems will continue to find interpretation challenging.
5. Conclusions
 We completed a large, systematic study of microbiological
contamination of a state’s groundwater resources with repeated
sampling of public wells.
 We observed widespread but sporadic fecal contamination us-
ing a comprehensive suite of pathogens and fecal markers, and
there were no obvious differences in contamination by system
type or geology.
 HF183 Bacteroides was detected frequently, but we cannot
directly assess health risk from the HF183 data; however, we
report the first assessment of its performance as an indicator of
pathogen presence in groundwater and show that it performed
similar to other microbial indicators.
 Analysis of many microorganisms by qPCR provided a compre-
hensive characterization of microbiological groundwater qual-
ity, but interpretation and application of such data remain a
challenge for regulators because the analytical capability has
outpaced the regulatory framework.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgements
We thank Dane Huber, Jared Schmaedeke, Trisha Sisto, Mike
Sutliff, and Nathan Gieske for well sampling. Funding was provided
by the Minnesota Clean Water, Land, and Legacy Amendment Fund.
The funding agency had no role in the study or its publication. Any
use of trade, firm, or product names is for descriptive purposes only
and does not imply endorsement by the U.S. Government. At the
time of publication, data have limited availability owing to
participating public water systems having the prerogative to
communicate raw water quality results. Contact corresponding
author for more information.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.watres.2020.115814.
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